Formulation optimization of functional wheat bread with low glycemic index from technological and nutritional perspective

Abstract Inclusion of prebiotic compounds as indigestible dietary fiber in wheat bread has grown rapidly considering the increased public awareness about their impact on health. However, through their incorporation, the technological characteristics may adversely be influenced by gluten dilution impacts. This study was done to evaluate the impacts of long chain, native and short chain inulin (L‐, N‐, and S‐type inulin, respectively) at 8%, 10%, 12%, 14%, and 16% w/w as Inulin Reconstituted Wheat Flour (IRWF) with similar gluten: carbohydrate ratio of wheat flour (at 10%, 12.5%, 15%, 17.5%, 20% w/w) on technological and nutritional value of wheat bread. Results indicated that despite no gluten dilution induced by IRWF supplementation, technological characteristics were adversely influenced especially at higher substitution level of L‐type‐containing formulations which is attributed to their higher water absorption index (WAI). Reversely, the nutritional value was positively influenced in which the lowest hydrolysis index (26.64%); predicted Glycemic Index (51.93%) and fructan loss content (25.42%) were found at L‐type inulin‐containing IRWF at the highest substitution level (20% w/w). As the nutritional value of wheat bread as staple foodstuff is important, optimizing the bread‐making process to decrease all reverse impacts induced by L‐inulin‐type inclusion seems to be required.

its digestibility ratio. In this regard, starch is classified as rapidly and slowly digestible starch abbreviated as RDS and SDS, respectively, and resistant starch (RS) (Chung et al., 2009). High incorporation of RS and SDS are claimed to have low GI (Bharath Kumar & Prabhasankar, 2014).
Regarding the high GI of white wheat bread as a staple foodstuff, any attempt to reduce the portion of carbohydrate and its digestibility is appreciated especially for people who suffer from obesity, diabetes, and insulin resistance. Using protein, fat and fiber are considered as the main strategies to reduce the GI of foods rich in carbohydrates like wheat bread (Wee & Henry, 2020). Considering the importance of carbohydrates in the technological characteristics determination of wheat bread, using dietary fiber is recommended to be useful in the formulation of low GI wheat bread.
Prebiotics as indigestible carbohydrates act as bifunctional agents. In other words, their incorporation in food formulations, however decreases their postprandial glucose response, and enhances the growth/activity of probiotic bacteria in the gastrointestinal tract (Carlson et al., 2017;Mohebbi et al., 2018). Among different compounds known as prebiotic, Inulin as a polymeric compound of fructose with a polymerization degree of 2-60, joined by β1-2 linkages is one of the most commonly used compounds in food formulations (Shoaib et al., 2016). Inulin is categorized considering its degree of polymerization (DP) as short chain (S) (DP ≤10), long chain (L) (DP ≥23), and native inulin (N) (DP: 2-60).
Inulin is found naturally in some plants, microorganisms or produced enzymatically from sucrose syrup using fructosyl transferases (Mollakhalili- . Regular intake of inulin as a prebiotic compound can stimulate the growth of bifidobacteria, produce short-chain fatty acids (SCFAs) and lower the intestinal pH, thus reducing the activity of pathogenic bacteria, cholesterol absorption, and colon cancer risk, alongside the reduction of GI in its incorporated food supply (Sirbu & Arghire, 2017). However, keeping the intact structure of inulin through food formulation and processing is prerequisite to be efficient as prebiotic; the technological and organoleptic characteristics of wheat bread should not be also negatively affected to guarantee its regular intake by the consumers. As the dietary fiber incorporation can adversely impact the technological characteristics of wheat bread through gluten dilution impact, reconstitution technique was used in this study. In other words, the quantities of starch and gluten were determined in wheat flour and used at the same ratio in Inulin Reconstituted Wheat Flour (IRWF) (Arya et al., 2016).
Considering the importance of polymerization degree of inulin in determination of its technological characteristics efficiency, digestibility and processing susceptibility, the aim of this study was to assess the impacts of inulin DP and IRWF incorporation level on technological and in vitro nutritional value of wheat bread.

| Method
This study with the aim of studying the potential of Inulin incorporation in reconstitution of wheat bread from technological characteristics and nutritional value perspective was set in full factorial experimental design. So, the impact of inulin DP (S, L, and N) and substitution level of inulin reconstituted wheat flour (IRWF) was investigated as demonstrated in Table 1. The proportion of gluten and inulin was similar to the gluten: starch ratio of basis wheat flour which is 15.33% w/w and 61.32% w/w, respectively.

| Farinograph
Brabender farinograph (Brabender, Germany) was used to determine the quantity of water to reach the consistency of 500 BU according to the AACC Method 54-21A. In this regard, the gluten and inulin were mixed well with wheat flour into the 300 g mixing bowl of farinograph which was operated at 30 ± 0.2°C. Farinograph curves were used for determination of the water absorption (percentage of water needed to provide a dough consistency of 500 BU), dough development time (time to reach a consistency of 500 BU), stability time (time in which the dough consistency is over 500 BU) and falling quality number were obtained.

| Preparation of bread
Bread preparation on the basis of formulations represented in Table 1 was done as follows with all ingredients added on flour dry basis. So, active dry yeast, sugar, salt, and canola oil at 2.2%, 0.5%, 1% and 3% w/w were mixed. The water inclusion level was on the basis of farinograph data calculation. The mixture was then stored inside the fermentation cabinet for 4 h at 29 ± 0.5°C. The baking process was done in a convection oven at 220 ± 10°C. Determination of proper baking time was according to feasibility of dough separation from the pan.

| Specific volume
Rape Seed displacement method was used for volume determination of loaves according to AACC 109 method 10-05. After volume and weight determination, specific volume was determined by their ratio as follows:

| Textural parameters
Instrumental texture profile analysis of breads was done and followed using texture profile analyzer (TA 20., KOOPA). Regarding, a 5 kg loading cell and cylinder probe (diameter of 43 mm), bread crumb with dimension of 20 × 20 × 25 mm was pressed up to its half height at speed ratio of 1 mm/s. The analysis was done in five replicates at room temperature. Texture profile analysis curves were assessed for hardness, springiness, and chewiness determination (Mohammadi et al., 2021).

| Crust and crumb color estimation
The Hunter Lab instrument was used for color determination of bread (both crust and crumb). Results are expressed as L* (brightness/darkness), a* (redness/greenness), and b* (yellowness/blueness) indices (Shiri et al., 2021).

| Sensory evaluation
Bread samples were sensory evaluated by nine-point hedonic scale.
In this regard, 1, 2, 3, 4, 5, 6, 7, 8, and 9 were indicators of extremely unpleasant, very much unpleasant, moderately unpleasant, slightly unpleasant, generally acceptable, slightly pleasant, moderately pleasant, very much pleasant and extremely pleasant bread samples, respectively. Color, flavor, texture, and overall acceptability were assessed as qualitative parameters by 30 semi-trained panelists (male: female ratio of panelists was 50:50 with the range of 18-58 years old). So, encoded bread samples were served randomly. The panelist's healthiness (with no allergy to gluten protein) and eagerness to be participated were also considered (Menon et al., 2015).
Regarding, 10 μl of sugar extract (by adding 1 ml of 0.005 M acetic acid to 50 mg bread sample, vortexing for 10 min and centrifuging at 12000 × g for 10 min [Figueroa & Khan, 1991]) was transferred to 5% aqueous solution of phenol (300 μl). After incubation for 5 min, 1.8 ml of concentrated sulfuric acid (18.4 M) was added. They were vortexed for 30 s and incubated in a water bath at room temperature for 20 min. Then, absorption was read at 490 nm. Total sugar was calculated as mg glucose/g extract.

| Resistant starch (RS)
The RS was estimated on the basis of the method described by (Reshmi et al., 2017). The protein removal was done by incubation of 100 mg sample with pepsin solution (40,000 U/ml, 1 g/10 ml KCl-HCl buffer) at 40°C for 60 min. Pancreatic α-amylase solution (40 mg a-amylase: 200 U/ml) was used for 16 h at 37°C to hydrolyze starch.
After washing with ethanol (99% v/v) and centrifuging, the supernatant pellets were digested with 2 M potassium hydroxide. Incubation of digested pellet and supernatant was done separately with amyloglucosidase (3300 U/ml) and the released glucose was measured by GODPOD kit and absorbance at 510 nm. The RS and Digestible starch (DS) were quantified as follows:

| Predicted glycemic index (pGI)
In vitro method was used to determine the pGI on the basis of the procedure proposed by (Reshmi et al., 2017). Regarding, 100 mg of ground bread sample was incubated with HCl-KCl buffer (10 ml and pH 1.5) and 200 μl pepsin solution (100 mg/ml HCl-KCl buffer) for 1 h at 40°C. By adding 200 μl pancreatic α-amylase solution (1.5 mg/10 ml phosphate buffer), the pH was increased to 7.8 and incubated for 45 min at 37°C. To stop the enzyme reaction, Na 2 CO 3 solution was added at 70 μl and using trismaleate buffer at pH = 6.9, samples were diluted to 25 ml. Then, 5 ml of pancreatic α-amylase solution (3 U/5 ml tris-maleate buffer) was added and incubation was done at 37°C.

| Fructan content quantification
The quantification of inulin in bread formulations was done enzymatically. In this regard, about 1 g bread sample was dissolved in 80 ml water (80°C). The mixture was filtered and treated with 1 ml sucrase/amylase solution (sucrase (50 U), β-amylase (500 U), pullulanase (100 U), and maltase (1000 U) in 22 ml sodium maleate buffer (pH 6.4) to remove sucrose, starch and reducing sugars). Afterward, alkaline borohydride (10 mg/ml sodium borohydride in 50 mM NaOH) was added and incubated at 40°C for 30 min and the excess was removed using acetic acid (100 mM) to reach the pH equal to 4.5. Quantification of fructan was done by fructanase (350 U/ml exo-inulinase and 35 U/ml endo-inulinase) for 20 min at 40°C. The fructan content (%) was estimated as: where: A: absorbance of 0.2 ml of the reactive solutions read against the reagent blank.
F: equal to 54.5, factor used to convert the absorbance value to μg fructose.
V: volume of used extractant.
M: weight of test portion.

| Statistical analysis
Quantitative characteristics were described using mean and standard deviation. All examinations were done in triplicate. Data analysis was performed using two-way anova and Kruskal-Wallis for parametric and non-parametric tests, respectively, by spss V.21 software at a significance level of 0.05.

| Effect of IRWF on farinograph parameters
The viscoelastic network formation of dough is influenced greatly by the other ingredients (Meybodi et al., 2019 Table 2. (2) RS: glucose (mg) × 0.9 As demonstrated, wheat flour supplementation with IRWF influenced the WA content in an inulin DP-dependent manner (p < .05). In other words, while the ability to absorb water is remained quite unaffected at formulation containing 8% w/w S-type inulin-containing IRWF (F1) (p > .05), it has been increased in the presence of 8% w/w L-type inulin-containing one (F10) compared to control (F16). The WA enhancement at formulations supplemented with IRWF-containing long chain inulin is mainly attributed to their ability to provide a gel-like structure potentially able to restore water as reported by (Mohammadi et al., 2021). Regardless of inulin DP, it is clear that supplementation level is also influential in WA quantity determination of wheat flour which is in the range of 2.05%-15.43%, 8.93%-17.66%, and 15.46%-23.53% w/w for S, N, and L inulin-containing IRWF (at ascending ratio), respectively. However, this enhancement is more obvious at longchain inulin-containing formulations. The WA enhancement via inclusion of different vegetable protein has also been previously stated which is attributed to their difference in WA capacity and consequently increased WA value in farinograph plot (Coţovanu & Mironeasa, 2022).
Dough development time (DDT) is also found to be influenced by IRWF supplementation. DDT is the required time for flour to be hydrated, developed, and provided consistent dough equal to 500 Brabender Unit (BU) consistencies. Results indicated significant (p < .05) enhancement of DDT at all IRWF-containing formula depending on polymerization degree of inulin and IRWF supplementation level (p < .05) ( Table 1). DDT enhancement induced by IRWF inclusion could be attributed to physicochemical characteristics difference in constituents of IRWF and wheat flour (Paraskevopoulou et al., 2010). Increased DDT (p < .05) found by increasing the DP of incorporated inulin in IRWF formulation, is also attributed to higher susceptibility of L-type inulin to interact with gluten proteins and prevent their hydration and development and consequently lead to DDT enhancement (Graça et al., 2020).
Stability time (ST) as stability indicator of dough is significantly influenced by dough constituents. As depicted, the lowest and highest ST are found in F16 (control) and F15 (long chain inulin-containing IRWF at 20% w/w) with contents equal to 8.73 and 19.23 min, respectively. In other words, wheat dough supplementation using IRWF enhanced the ST significantly on the basis of its supplementation level and polymerization degree of incorporated inulin. In this regard, it seems that dough samples with higher IRWF incorporation level had higher ST and consequently mixing resistance compared to the control sample. Previously, it has been stated that differently originated proteins in dough mixtures are not fully hydrated and behaved as dispersed particles which reinforce the gluten network and consequently increase their derived dough ST (Paraskevopoulou et al., 2010). Table 2, the farinograph quality number (FQN) which is an indicator of gluten protein quality and quantity is enhanced by IRWF supplementation level and inulin DP. TA B L E 2 Farinograph characteristics of wheat flour supplemented with inulincontaining reconstituted wheat flour

| Effects of IRWF on physicochemical characteristics
The impacts of IRWF supplementation on physicochemical characteristics of wheat breads have been investigated and represented in Table 3.
As depicted, specific volume (SV) is significantly influenced by supplementation level and the type of incorporated inulin. In other words, despite no significant difference between F16 (Control sample) and F1 (containing S inulin type and supplementation level at 10% w/w) (p ≥ .05), about 48.30% w/w decreased is found in SV of F15 compared to control sample (p < .05) at. As SV is affected by dough strength and its prospective in restoring gases through bread-making (Kou et al., 2019;Whitney & Simsek, 2020), this reduction in IRWF supplemented formulations may be induced by dough elasticity reduction provoked by their weakening impacts on gluten network. However, no gluten dilution is observed in all supplemented formulations, it seems more than early fixing of structure in L-type inulin-containing IRWF formulation induced by their higher water content leads to retardation in the development of gluten network (Sabanis & Tzia, 2009). In other words, higher affinity of L-type inulin to bound water resulted in its competence with gluten and consequently reduce its development and ability to restore gases produced through fermentation process. This finding is in accordance with (de Erive et al., 2020).
The textual characteristics of IRWF-supplemented-wheat breads in terms of hardness, chewiness, and springiness are demonstrated in Table 3. As depicted, the highest and lowest hardness is found in F16 and F15 at 756.09 and 3189.11 g, respectively. Changes in chewiness characteristics are also in similar order of harness. In other words, the crumb hardness and chewiness have been increased by IRWF supplementation level and polymerization degree of incorporated inulin (p < .05). Considering the impact of inulin on crumb hardness, however, it has previously stated that its incorporation in wheat bread provides a coarse crumb structure with higher hardness (Frutos et al., 2008;Sardabi et al., 2021;Sirbu & Arghire, 2017), its adverse impact has been found at this study which is improved by increasing the inulin degree of polymerization. On the other hand, despite decreased hardness and increased SV via gluten protein inclusion in steamed bread (Qian et al., 2019), a reverse order has been found at this study which is supposed to be induced by more coarse crumb structure formed by formulations replaced by IRWF at high level, especially those containing long-chain inulin as demonstrated by (Mohammadi et al., 2021). As a less elastic system is formed through IRWF supplementation, it is also declared by springiness data. Springiness as an indicator of deformation ratio of a deformed product to its un-deformed state after removal of deforming force is directly dependent on hardness and chewiness. Generally, any variation in formulation will be corresponded in springiness and chewiness differentiation (Eriksson et al., 2014). Increasing the springiness value denote a well-developed gluten network with more elasticity in crumb. As demonstrated in

TA B L E 3
Physicochemical characteristics of wheat breads supplemented with inulin-containing reconstituted wheat flour for control and F16 samples, respectively. The decrease observed in springiness characteristics of IRWF-incorporated wheat bread formulations containing L-type inulin could be attributed to their lower strength and gas retention capability of gluten network. However, incomplete starch swelling through baking will also restrict the sponge-like formation in bread crumb. This result is verified (de Erive et al., 2020;Londono et al., 2015).

| Effects of IRWF on color parameters
Color characteristics are considered to be influential in consumer's acceptance determination of bread alongside its textural and physicochemical characteristics. Two mechanisms of Maillard and caramelization reactions are simultaneously evolved in color development of bakery products like wheat bread (Anil, 2007). The crust and crumb color determination of IRWF-supplemented wheat bread has been conducted by CIE L*a*b* color scale as represented in Table 4. The crust and crumb color are generally developed by Maillard/caramelization reactions and inherent color characteristics of formulation, respectively (Dhen et al., 2018). Crumb color is also influenced by structure forming characteristics of formulation through breadmaking process (NithyaBalaSundari et al., 2020).
As demonstrated in Table 4, IRWF-supplemented bread had significantly (p < .05) lower L*, and higher a* and b* in crust compared to the control bread. Considering the polymerization degree of inulin, the highest variation is found in S-type inulin samples. In other words, the lowest L* value is found in F5 at 72.18. The lightness reduction via low DP inulin incorporation has been previously stated by (Aguiar et al., 2021) which was supposed to be induced by their more susceptibility to be involved in Millard reaction. Decreased L* along with increased a* of wheat bread crust was previously related to increased Millard reaction ratio by (de Erive et al., 2020;Skendi et al., 2010). As the trend of changes in crust L* is inconsistent with a* and b* values, it seems that the yellow-red shade of proteins in crust color determination is also influensive (de Erive et al., 2020).
As mentioned previously, the crumb color characteristics of bread samples are influenced by their formulation and structure-forming characteristics (Kou et al., 2019). As demonstrated crumb color characteristics of wheat bread is influenced by its supplementation level and polymerization degree of incorporated inulin (p < .05). In other words, the highest and lowest L* are found in F15 (supplemented by 20% w/w L-type inulin-containing IRWF) and F5 (supplemented by 20% w/w S-type inulin-containing IRWF) with values at 79.12 and 89.15, respectively. It seems that higher susceptibility of S-type inulin to be fermented by yeast leads to more release of carbon dioxide and air bubble expanding through bread-making process which resulted in more light scattering and consequently decreased L* value.
Increased L* in F15 is accompanied by the highest b* and harness values which are in accordance with (Matos & Rosell, 2013).

| Sensory evaluation of IRWF supplemented wheat bread
Considering the importance of the overall acceptability of new products to be introduced in market, sensory assessment of IRWFsupplemented wheat bread was done and reported in Figure 1. Sensory characteristics were evaluated at four criteria of texture, flavor, color, and overall acceptability. As depicted, all samples are higher than mediate at all evaluated criteria. Generally, the highest score considering flavor, color, texture, and overall acceptability has been gained by F5 (containing S-type inulin at IRWF supplemented level of 20% w/w). As the specific volume and crust color are the main indicators to meet the expectative of consumers (Di Monaco et al., 2015;Pasqualone et al., 2019), the optimization is found in F5.

TA B L E 4 Crust and crumb color analysis of wheat breads supplemented with inulin-containing reconstituted wheat flour
Considering the flavor sensation, the highest score is found in samples containing S-type inulin which can be attributed to higher metabolites of Millard reaction as described by (Mohammadi et al., 2021). The overall acceptability is well matched with texture characteristics (r = 0.87, p < .05). formulations. The decrease in TS seems to be more pronounced at formulations containing lower DP inulin. As the lower DP inulin is more susceptible to be hydrolyzed through bread making process, it will be more remarkably lost by Millard reaction through baking by Millard reaction (Morreale et al., 2019). However, this decrease will be reversely influenced by increasing the level of IRWF incorporation. The increased Millard reaction ratio through S-type inulin incorporation is also obvious in crust color consideration.

| Nutritional value
The highest RS value is found in F15 containing the highest IRWF supplementation level provided by higher DP inulin with amount at 10.19% which simultaneously has the lowest DS of 58.12%.
Despite similar inulin incorporation level at different formulations, the difference in the rate of digestion seems to be induced by the differences in their nature and impacts on starch gelatinization. As the inulin gel formed at lower temperature, it prohibits the water accessibility for the amorphous region of starch and consequently restricts its gelatinization (Luo et al., 2017;Reshmi et al., 2017).
Protein inclusion is also reported to reduce the starch susceptibility to enzymatic attack by decreasing the starch granule swelling and its gelatinization (Graça et al., 2021). In this regard, increased RS has been found in all formulations by increasing the level of IRWF.
Hydrolysis indices (HI) which is calculated by comparison of glucose released from sample and standard glucose through the digestion process and its corresponding pGI is considerably influenced by the supplementation process. As depicted in Table 4, the highest and lowest HI is found at F16 and F15 with values at 57.08% and 22.6%, respectively, with simultaneously the highest and lowest pGI. In other words, increasing the polymerization degree of incorporated inulin significantly (p < .05) decreased the HI which are also decreased more sharply at formulations containing higher IRWF supplementation level. The decreased HI induced by increasing the level of IRWF and polymerization degree of incorporated inulin is attributed to decreased starch gelatinization ratio and its preventative impacts on hydrolyzing enzymes (Englyst et al., 2007;Garsetti et al., 2005;Gourineni et al., 2017).
Inulin is incorporated as a functional ingredient in this study through its prebiotic role. Consequently, its stability through bread making process is considerably important, as influensive prebiotics needs to be entered intact to upper intestinal tract. As the incorporation level of inulin was different, monitoring the fructan loss in IRWF-supplemented formulations indicate a progressive fructan loss through processing in the range of 25.42%-43.01% w/w.
In other words, the highest and lowest fructan loss is found in F15 and F1, respectively, which indicate the enhanced prebiotic loss by decreasing its incorporation level and polymerization degree. The enhanced inulin loss by decreasing its polymerization degree is attributed to its higher susceptibility to be used by yeast and take part in Millard reaction. This finding is in accordance with (Mohammadi et al., 2021).

| CON CLUS ION
Inulin supplementation of wheat bread in the form of inulin reconstituted wheat flour (IRWF) seems to be applicable from nutritional perspective, especially at formulations containing long-chain inulin type. However, despite gluten inclusion at formulations to conquer gluten dilution impacts, adverse impacts on technological characteristics are still restricting in industrially production of them especially long-chain inulin-containing ones which are attributed to their higher water absorption content, accelerated gluten network formation, and decreased starch gelatinization. However, long-chain inulin incorporation significantly improves the nutritional characteristics of wheat considering resistant starch content, hydrolysis index, predicted Glycemic index, and fructan loss parameters. Consequently, whole bread-making process as the type of fermentation, time: temperature ratio of fermentation process and increasing ratio of temperature through baking process should be optimized to formulate optimized formulation from both nutritional and technological perspective.

ACK N OWLED G M ENT
The financial support of this study is achieved by the Shahid Sadoughi University of medical science, Yazd, Iran.

CO N FLI C T O F I NTE R E S T
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm the availability of all data supporting the findings within the article.

E TH I C S S TATEM ENT
The Ethical aspects of this study are evaluated and approved by the